Kerry M. Hanson

Hybrid Molecule-Nanocrystal Photon Upconversion Across the Visible and Near-Infrared.

The ability to upconvert two low energy photons into one high energy photon has potential applications in solar energy, biological imaging, and data storage. In this Letter, CdSe and PbSe semiconductor nanocrystals are combined with molecular emitters (diphenylanthracene and rubrene) to upconvert photons in both the visible and the near-infrared spectral regions. Absorption of low energy photons by the nanocrystals is followed by energy transfer to the molecular triplet states, which then undergo triplet-triplet annihilation to create high energy singlet states that emit upconverted light. By using conjugated organic ligands on the CdSe nanocrystals to form an energy cascade, the upconversion process could be enhanced by up to 3 orders of magnitude. The use of different combinations of nanocrystals and emitters shows that this platform has great flexibility in the choice of both excitation and emission wavelengths.

Observation and Quantification of Ultraviolet‐induced Reactive Oxygen Species in Ex Vivo Human Skin¶

Two‐photon fluorescence imaging is used to detect UV‐induced reactive oxygen species (ROS) in ex vivo human skin in this study. ROS (potentially H2O2, singlet oxygen or peroxynitrite [or all]) are detected after reaction with nonfluorescent dihydrorhodamine‐123 (DHR) and the consequent formation of fluorescent rhodamine‐123 (R123). The cellular regions at each epidermal stratum that generate ROS are identified. R‐123 fluorescence is detected predominately in the lipid matrix of the stratum corneum. In contrast, the strongest R123 fluorescence signal is detected in the intracellular cytoplasm of the viable epidermal keratinocytes. A simple bimolecular one‐step kinetic model is used for estimating the upper bound of the number of ROS that are generated in the skin and that react with DHR. After ultraviolet‐B radiation (280–320 nm) (UVB) equivalent to 2 h of noonday summer North American solar exposure (1600 J m−2 UVB), the model finds that 14.70 × 10−3 mol of ROS that react with DHR are generated in the stratum corneum of an average adult‐size face (258 cm−2). Approximately 10−4 mol are potentially generated in the lower epidermal strata. The data show that two‐photon fluorescence imaging can be used to detect ROS in UV‐irradiated skin.

Application of Nonlinear Optical Microscopy for Imaging Skin

Recent advances in the use of nonlinear optical microscopy (NLOM) in skin microscopy are presented. Nonresonant spectroscopies including second harmonic generation, coherent anti‐Stokes Raman and two‐photon absorption are described and applications to problems in skin biology are detailed. These nonlinear techniques have several advantages over traditional microscopy methods that rely on one‐photon excitation: intrinsic 3D imaging with <1 μm spatial resolution, decreased photodamage to tissue samples and penetration depths up to 1000 μm with the use of near‐infrared lasers. Thanks to these advantages, nonlinear optical spectroscopy has become a powerful tool to study the physical and biochemical properties of the skin. Structural information can be obtained using the response of endogenous chemical species in the skin, such as collagen or lipids, indicating that optical biopsy may replace current invasive, time‐consuming traditional histology methods. Insertion of specific probe molecules into the skin provides the opportunity to monitor specific biochemical processes such as skin transport, molecular penetration, barrier homeostasis and ultraviolet radiation‐induced reactive oxygen species generation. While the field is quite new, it seems likely that the use of NLOM to probe structure and biochemistry of live skin samples will only continue to grow.

Two-Photon Fluorescence Imaging and Reactive Oxygen Species Detection Within the Epidermis

Two-photon fluorescence microscopy is used to detect ultraviolet-induced reactive oxygen species (ROS) in the epidermis and the dermis of ex vivo human skin and skin equivalents. Skin is incubated with the nonfluorescent ROS probe dihydrorhodamine, which reacts with ROS such as singlet oxygen and hydrogen peroxide to form fluorescent rhodamine-123. Unlike confocal microscopic methods, two-photon excitation provides depth penetration through the epidermis and dermis with little photodamage to the sample. This method also provides submicron spatial resolution such that subcellular areas that generate ROS can be detected. In addition, comparative studies can be made to determine the effect of applied agents (drugs, therapeutics) upon ROS levels at any layer or cellular region within the skin.

Applications of ultrafast lasers to two-photon fluorescence and lifetime imaging

Fluorescent probes have found widespread use in biomedical sciences. Particularly since they can be targeted to cellular compartments and further more can report on the properties of their environment such as calcium concentration. Near infrared ultrafast lasers find increasing use for fluorescence applications since femtosecond pulses with a few milliwatts of average power are sufficient to induce significant two photon fluorescence from the probe when focused into typical samples. The nonlinear optical excitation process allows sectioned imaging of 3-D samples without use of a confocal pinhole. In this paper we describe two aspects of multiphoton microscopy: the two- photon excitation cross section and the fluorescence lifetime. Of interest is the wavelength characterization of two-photon excitation cross-sections of fluorescence probes. We slowly modulate (~500Hz) the intensity envelope of the input laser pulse train and analyze the emission signal in terms of the amplitude and phase of the harmonics of this modulation. In effect this is a power study that allows separation of different order effects. An application of ultrafast laser excitation that exploits many of the features outlined above is measurement of pH gradients in the skin. This is essential to skin barrier function and disruption of the gradient is thought to be an indicating factor in many skin diseases. A probe for which the fluorescence lifetime varies with pH is used. We thus are able to tackle problems associated with inhomogeneous labeling. We have developed a two-photon laser-scanning lifetime microscope and present pH maps of skin obtained with this instrument.

Two-photon standing-wave fluorescence correlation spectroscopy

A fluorescence correlation spectroscopy experiment that combines two-photon excitation and a standing-wave interference pattern is presented. The experimental correlation function can be analyzed using a simple expression involving (1) an exponential decay with time constant tau(f), which reflects diffusion across the interference fringes, and (2) a longer-lived decay with time constant tau(omega), which reflects diffusion in and out of the focal spot. The diffusion of Rhodamine 110 in water and ethylene glycol is measured using this method. The ability to simultaneously measure diffusion on two different time and lengthscales makes this experiment especially useful in environments where anomalous diffusion is suspected.

Augmenting Skin Photoprotection Beyond Sunscreens

Sun exposure generates an abundance of reactive oxygen species (ROS) within skin, which overwhelm skin’s natural defenses (leading to oxidative stress) and which over the course of our lives exact a toll on skin’s health and appearance (especially photoaging).

Two-Photon Standing Wave Microscopy to Measure Small Scale Motions

A two-photon standing wave fluorescence experiment is used to look at the small-scale motions of fluorescently-labeled DNA in live cells and biological systems. Both photobleaching and fluorescence correlation spectroscopy versions of this experiment are presented.